Interaction of diphtheria toxin with mammalian cell membranes

Uptake of 125I-labeled diphtheria toxin and serologically related proteins by a sensitive human HeLa cell line and by a resistant mouse L929 cell line has been studied. The evidence suggests that there is an initial rapid reaction between a recognition site present on the toxin Fragment B and specific plasma membrane receptors on the sensitive cell (there are approximately 4000/HeLa cell). This initial interaction is followed by a slow irreversible process during which there is a major conformational alteration of the toxin molecule causing the enzymically active 22,000-dalton Fragment A to become exposed to the cytosol. We suggest that it is at this point that cleavage of the NH2-terminal disulfide bond occurs leading to release of Fragment A into the cytoplasm. The toxin Fragment B remains attached to the membrane, probably formed in a complex with receptor, and blocks entry of additional toxin molecules through the same site. Specific membrane receptors are lacking from mouse cells. Both HeLa cells and L929 cells internalize toxin, related nontoxic proteins, and inert molecules such as inulin nonspecifically into endocytotoc vesicles. At 30 degrees the bulk internalization of extracellular fluid is about 1.2% of their cell volume per h for both cell lines. Fragment A does not traverse the plasma membrane by a mechanism that depends on endocytosis. The interaction of diphtheria toxin with the sensitive cell membrane is discussed in relation to other protein toxins and certain glycopeptide tropic hormones in which relatively large, hydrophilic polypeptide fragments or subunits are presumed to traverse the target cell plasma membrane and reach the cytoplasm in biologically active form.